ß-Strand-mediated Domain-swapping in the Absence of Hydrophobic Core Repacking.

Domain swapping is a process wherein a portion of a protein is exchanged with its counterpart in another copy of the molecule, resulting in the formation of homo-oligomers with concomitant repacking of a hydrophobic core. Here, we report domain swapping triggered upon modifying a ß-hairpin sequence within a single-layer ß-sheet (SLB) of a model protein, OspA that did not involve the formation of a reorganized hydrophobic core. The replacement of two ß-hairpin sequences with a Gly-Gly and shorteing of a ß-hairpin resulted in a protein that formed two distinct crystal structures under similar conditions: one was monomeric, similar to the parental molecule, whereas the other was a domain-swapped dimer, mediated by an intermolecular ß-sheet in the SLB portion. Based on the dimer interface structure, we replaced the Gly-Gly sequence with three-residue sequences that enable the formation of a consecutive intermolecular ß-sheet, including the Cys-Thr-Cys sequence that formed a stable disulfide-linked dimer. These results provide new insights into protein folding, evolution, and the designability of protein structure.

The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth.

Newts can regenerate their limbs throughout their life-span. Focusing on muscle, certain species of newts such as Cynops pyrrhogaster dedifferentiate muscle fibers in the limb stump and mobilize them for muscle creation in the regenerating limb, as they grow beyond metamorphosis. However, which developmental process is essential for muscle dedifferentiation, metamorphosis or body growth, is unknown. To address this issue, we tracked muscle fibers during limb regeneration under conditions in which metamorphosis and body growth were experimentally shifted along the axis of development. Our results indicate that a combination of metamorphosis and body growth is necessary for muscle dedifferentiation. On the other hand, ex vivo tracking of larval muscle fibers revealed that newt muscle fibers have the ability to dedifferentiate independently of metamorphosis and body growth. These results suggest that newt muscle fibers have an intrinsic ability to dedifferentiate, but that metamorphosis and body growth are necessary for them to exhibit this hidden ability. Presumably, changes in the extracellular environment (niche) during developmental processes allow muscle fibers to contribute to limb regeneration through dedifferentiation. This study can stimulate research on niches as well as gene regulation for dedifferentiation, contributing to a further understanding of regeneration and future medical applications.

Surface Engineering of Top7 to Facilitate Structure Determination.

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular ß-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular ß-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

Structural analysis of the ß-sheet edge of peptide self-assembly using a model protein.

Peptides and proteins self-assemble into ß-sheet-rich fibrils, amyloid, which extends its structure by incorporating peptide/protein molecules from solution. At the elongation edge, the peptide/protein molecule binds to the edge of the amyloid ß-sheet. Such processes are transient and elusive when observing molecular details by experimental methods. We used a model protein system, peptide self-assembly mimic (PSAM), which mimics an amyloid-like structure within a globular protein by capping both edges of single-layer ß sheet (SLB) with certain domains. We constructed a PSAM variant that lacks the capping domain on the C-terminal side to observe the structure of the ß-sheet edge of the peptide self-assembly. This variant, which we termed PSAM-edge, proved to be soluble with a monomeric form. Urea-induced unfolding experiments revealed that PSAM-edge displayed two-state cooperative unfolding, indicating the N-terminal capping domain and extended SLB folded as one unit. The crystal structure showed that SLB was almost completely structured except for a few terminal residues. A molecular dynamics simulation results revealed that the SLB structure was retained while the C-terminal four residues fluctuated, which was consistent with the crystal structure. Our findings indicate that SLB is stable even when one side of the ß-sheet edge is exposed to a solvent. This stability may prevent the dissociation of the attached peptide from the peptide self-assembly. Because of the scarcity of SLB proteins with exposed ß-sheet edges in nature, successful construction of the PSAM-edge expands our understanding of protein folding and design.

Cooperative unfolding of a single-layer ß-sheet protein, CPAP G-box.

CPAP is a centriolar protein and its C-terminal domain, G-box or TCP, has a very unique structure that comprises a single-layer ß-sheet without hydrophobic core packing. Here we characterized its biophysical properties, including its stability against chemical denaturation. Interestingly, upon urea-induced equilibrium unfolding, the CPAP G-box showed cooperative unfolding behavior that is the hallmark of globular proteins. We analyzed the m-value, a measure of the cooperative transition, from the urea-induced unfolding and found that the estimated m-value from surface burial upon folding is consistent with the experimental value, supporting the two-state unfolding. Next, we constructed deletion mutants of the terminal ß-strands and found that the mutants showed reduced stability. The unique structure and characteristics of CPAP G-box provides an interesting opportunity to observe how the core-less flat ß-sheet protein can be folded in solution.

Crystal structure of the catalytic unit of GH 87-type α-1,3-glucanase Agl-KA from Bacillus circulans.

Glycoside hydrolase (GH) 87-type α-1,3-glucanase hydrolyses the α-1,3-glucoside linkages of α-1,3-glucan, which is found in fungal cell walls and extracellular polysaccharides produced by oral Streptococci. In this study, we report on the molecular structure of the catalytic unit of GH 87-type α-1,3-glucanase, Agl-KA, from Bacillus circulans, as determined by x-ray crystallography at a resolution of 1.82 Å. The catalytic unit constitutes a complex structure of two tandemly connected domains-the N-terminal galactose-binding-like domain and the C-terminal right-handed ß-helix domain. While the ß-helix domain is widely found among polysaccharide-processing enzymes, complex formation with the galactose-binding-like domain was observed for the first time. Biochemical assays showed that Asp1067, Asp1090 and Asp1091 are important for catalysis, and these residues are indeed located at the putative substrate-binding cleft, which forms a closed end and explains the product specificity.

Domain-Swapping Design by Polyproline Rod Insertion.

During domain swapping, proteins mutually interconvert structural elements to form a di-/oligomer. Engineering this process by design is important for creating a higher order protein assembly with minimal modification. Herein, a simple design strategy is shown for domain-swapping formation by loop deletion and insertion of a polyproline rod. Crystal structures revealed the formation of the domain-swapped dimers and polyproline portion formed a polyproline II (PPII) structure. Small-angle X-ray scattering demonstrated that an extended orientation of domain-swapped dimer was retained in solution. It is found that a multiple of three of inserting proline residue is favored for domain swapping because of the helical nature of PPII. The rigid nature of the polyproline rod enables precise control of the interdomain distance and orientation.